EP3295926A1 - Exopolysaccharid-protein-komplex, verfahren zur vorbereitung des komplexes und verwendungen davon - Google Patents

Exopolysaccharid-protein-komplex, verfahren zur vorbereitung des komplexes und verwendungen davon Download PDF

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EP3295926A1
EP3295926A1 EP16382436.0A EP16382436A EP3295926A1 EP 3295926 A1 EP3295926 A1 EP 3295926A1 EP 16382436 A EP16382436 A EP 16382436A EP 3295926 A1 EP3295926 A1 EP 3295926A1
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Prior art keywords
exopolysaccharide
protein complex
halomonas
composition
cancer
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French (fr)
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Víctor Manuel Deroncelé Thomas
Rafael Montilla Arevalo
Josep Castells Boliart
Adrián GARCÍA DE LA MARINA
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Institut Universitari de Ciencia i Tecnologia
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Institut Universitari de Ciencia i Tecnologia
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Priority to EP16382436.0A priority Critical patent/EP3295926A1/de
Priority to US16/334,346 priority patent/US11253457B2/en
Priority to EP17842382.8A priority patent/EP3544582A1/de
Priority to PCT/EP2017/073606 priority patent/WO2019029833A1/en
Publication of EP3295926A1 publication Critical patent/EP3295926A1/de
Priority to US17/570,048 priority patent/US20220160610A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/06—Preparations for care of the skin for countering cellulitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to the field of biotechnology.
  • the present invention relates to an exopolysaccharide-protein complex obtained from bacteria.
  • the present invention further relates to a method of preparing said complex, a composition comprising thereof and uses thereof.
  • EPS Microbial exopolysaccharides
  • EPS production is among the biochemical strategies used by microorganisms present in hypersaline environments, in order to survive in high saline conditions.
  • the genus Halomonas has received increasing interest as several species are able to produce significant quantities of EPS with high surface activity and/or have rheological properties and applications in food, cosmetic and pharmaceutical sectors (Poli, Anzelmo, & Nicolaus, 2010).
  • Microbial EPS are biopolymers with a high molecular weight having an extreme diversity in terms of chemical structure and composition.
  • Polysaccharides are the most abundant component of the EPS but previous electron microscopy studies (Nevot, Deroncele, Lopez-Iglesias, et al., 2006; Nevot, Deroncele, Messner, Guinea, & Mercade, 2006), heavily emphasized that other macromolecules such as proteins can also be present.
  • EPS also offer some important biomedical properties, including antitumor activities (Bazani Cabral de Melo et al., 2015; Ye et al., 2016), antimutagenicity (Miranda et al., 2008), anti-ulcer (Rasulov et al., 1993), anti-inflammatory properties and immune-modulating activities (Ciszek-Lenda, Nowak, Srottek, Gamian, & Marcinkiewicz, 2011).
  • EPS Anti-inflammatory and immune-modulating activities of EPS are drawing much attention and in lactic acid bacteria were related to the physicochemical properties and structural characteristics of their EPS (Gorska et al., 2014; Shao et al., 2014; Yasuda, Serata, & Sako, 2009).
  • An important mechanism involved in the immunostimulatory activity of polysaccharides is their ability to enhance macrophage function (Beutler, 2004). Polysaccharides were reported to be the active immunomodulators that potentiate both innate and adaptive immunity.
  • NF- ⁇ B a transcription factor that promote the expression of variety of molecules involved in immune, inflammatory and acute phase responses, including NO and tissue necrosis factor alpha (Li et al., 2015), plays an important role in this activation process.
  • chemopreventive activity of polysaccharides is based on their tumor anti-initiating activity through their modulation of carcinogen metabolism, in addition to the tumor anti-promoting activity through their anti-inflammatory activity (Gamal-Eldeen, Ahmed, & Abo-Zeid, 2009; Raafat, Gamal-Eldeen, El-Hussieny, Ahmed, & Eissa, 2014).
  • US 7348420 B2 describes the recombinant expression of cell wall, cell surface, and secreted proteins of Lactobacillus acidophilus potentially useful for the treatment or prevention of cancer, particularly colon cancer. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use.
  • US 8129518 B2 provided synthetic polysaccharide antigens with anti-inflammatory or inflammatory immunomodulatory properties.
  • this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use.
  • the synthetic polysaccharide is composed mainly of lipopolysaccharide, and bacterial cell wall glycopeptides, also known as murein or peptidoglycan, from both Gram negative and Gram positive bacteria.
  • US 8088605 B2 discloses a delivery system for active molecule comprising exopolysaccharide micelles produced by a Lactobacillus strain. These includes active molecules such as DNA, RNA, protein, peptide, peptidomimetic, virus, bacteria, nutraceutical product and pharmaceutical agent with analgesic, anesthetic, antibiotic, anticancer, anti-inflammatory, and antiviral properties. However, these pharmaceutical agents are not produced by the bacterium.
  • WO 2009/127057 A1 discloses a skin care composition comprising one exopolysaccharide derived from a microbial mat. This document discusses the use of different compositions for cosmetic or therapeutic approaches and provides examples for evaluating the effects of the EPSs on the synthesis of hyaluronic acid, lipid synthesis, among others, related with cosmeceuticals applications. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use as anti-inflammatory or immunomodulatory agents.
  • CN 104694594 discloses a preparation method of a sea cucumber epiphytic Bacillus subtilis exopolysaccharide that can be used in the anti-tumor medicines, cosmetic additives and other fields.
  • the principal application exposed in this document is related with the application of this EPS in bacterial and plant pathogen growth inhibition, a peroxide scavenger, anti-tumor and inhibition of microapplication.
  • US9095733 discloses methods of using polysaccharides for applications in topical personal care products, cosmetics, and for wrinkle reduction compositions. Particularly, this document provides compositions of microalgal cells with high value cosmeceutical ingredients such as carotenoids, polyunsaturated fatty acids, moisturizing polysaccharides, superoxide dismutase, and other components. However, this document does not disclose the combination of extracellular polysaccharides and selected associated-proteins and its use as anti-inflammatory or immunomodulatory agents.
  • WO 2013/082915 A1 discloses a strain of exopolysaccharide-secreting Lactobacillus brevis, and its applications in medicaments, healthcare products and food products for immunity enhancement.
  • the exopolysaccharide extract is obtained after precipitating protein via trichloroacetic acid method to remove the protein.
  • an exopolysaccharide-protein complex secreted by bacteria exhibits unique immunomodulating properties, is non-cytotoxic and non-proliferating to normal cell lines.
  • the exopolysaccharide-protein complex ameliorates pro-inflammatory chemokines expression and induces the production of other anti-inflammatory cytokines of cells in culture. It has the activity of inhibiting growth of tumor cell lines. Accordingly, the complex can be used for treatment or prevention of diseases in which inflammation and immunomodulation are critical or as adjuvant in medical treatment, such as those related to an imbalance of the production of anti-inflammatory or proinflammatory cytokines, and also for preparing anti-tumor drugs.
  • a first object of the invention relates to an exopolysaccharide-protein complex obtained from a bacterium comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins.
  • a second object of the invention relates to a method of preparing an exopolysaccharide-protein complex according to the first object.
  • a third object of the invention relates to a composition (pharmaceutical, nutraceutical or cosmeceutical) comprising an exopolysaccharide-protein complex according to the first object.
  • a fourth object of the invention relates to the use of the composition according to the third object of the invention and the exopolysaccharide-protein complex according to the first object of the invention.
  • isolated should be considered to mean material removed from its original environment in which it naturally occurs, for example, a bacterial strain from hypersaline environment.
  • EPS or "exopolysaccharide” or “EPSx” should be understood to mean high molecular weight polymers that are composed of sugar residues and expressed by bacteria.
  • activation of NF- ⁇ B means the process by which stimulation of NF- ⁇ B mediated by Toll-like receptors activates NF- ⁇ B, subsequently facilitating increased transcription of mRNA coding for intracellular production of particular chemokines and cytokines and subsequent translation of the transcribed mRNA, resulting in increased amounts of particular cytokines and chemokines that are both present intracellularly and released by the eukaryotic cell into the intercellular environment.
  • interleukin means any of a group of cytokines (secreted signaling molecules) that were first seen to be expressed by white blood cells. Interleukins are commonly designated using an abbreviation: e.g. IL-6, IL-8, etc.
  • immunomodulatory refers to its ability to modulate the response of cells of the human immune system.
  • anti-inflammatory refers to the ability to induce the production of interleukin-10, a potent anti-inflammatory cytokine and to block the production of interleukin 12, a cytokine pro-inflammatory nature.
  • cancer and “tumor” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • anticancer activity refers to preferential cytotoxic effect against tumor cells without any significant adverse effects to normal cells under the same conditions of exposure.
  • the term “nutraceutical” refers to any compounds or chemicals that can provide dietary or health benefits when consumed by humans or animals.
  • composition refers to a composition that is employed as both a cosmetic composition and as a pharmaceutical composition.
  • adjuvant refers to an embodiment of the invention provided to a subject in conjunction with a medical treatment plan.
  • immunomodulation refers to the ability to modify the immune responses in a subject in a way that may have healthful benefits, such as to produce an anti-inflammatory or an immunostimulatory effect.
  • RT-PCR means reverse transcription polymerase chain reaction (RT-PCR), a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule.
  • RNA ribonucleic acid
  • RT-PCR reverse transcription polymerase chain reaction
  • the RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process.
  • the present invention relates to an exopolysaccharide-protein complex (also called herein EPCx) obtained from a bacterium, preferably wherein said bacterium is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus, more preferably from genus Halomonas, still more preferably from Halomonas elongata sp., comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins.
  • EPCx exopolysaccharide-protein complex
  • exopolysaccharide-protein complex are not limited to (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins.
  • said exopolysaccharide-protein complex obtained from a bacterium consists of: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins.
  • the term "consists of” should be understood as the components of the exopolysaccharide-protein complex are exclusively (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins.
  • the exopolysaccharide-protein complex of the present invention relates to an exopolysaccharide-protein complex comprising: (i) a crude exopolysaccharide, and (ii) exopolysaccharide-associated proteins, wherein the crude exopolysaccharide is a heteropolymer comprising or consisting of:
  • the exopolysaccharide-protein complex of the invention comprises or consists of:
  • the crude exopolysaccharide further comprises sulfate, preferably at a concentration from 2 to 10 wt %.
  • the exopolysaccharide-associated proteins include two bands of molecular weight as obtained by SDS-PAGE.
  • the EPCx has a protein profile comprising at least, according to the SDS-PAGE technique, 12 detectable bands, including two principal bands, corresponding, respectively, to molecular weights (approximate molecular weights given in relation to molecular standards, notably provided by Bio-Rad Laboratories) ranging between:
  • the present invention relates to a method of preparing an exopolysaccharide-protein complex according to the first aspect of the invention, including each of the embodiments comprised in said first aspect and combinations thereof.
  • the method of preparing an exopolysaccharide-protein complex comprises the steps of:
  • the genus is selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus.
  • said bacteria genus is Halomonas and in a more preferred embodiment, Halomonas elongata sp. is used as a bacterium belonging to Halomonas genus.
  • the medium suitable to cultivate the above mentioned bacteria includes a synthetic medium comprising a carbon source selected from the group consisting of: lactose, maltose, glucose, galactose, sucrose, glycerol and mixtures thereof.
  • a carbon source selected from the group consisting of: lactose, maltose, glucose, galactose, sucrose, glycerol and mixtures thereof.
  • the carbon source is selected from the group consisting of: glucose, lactose, sucrose, and mixtures thereof.
  • the carbon source is lactose.
  • the other fermentation medium components are: potassium phosphate dibasic (K 2 HPO 4 ), 0.5 -1.0 wt%; potassium phosphate monobasic (KH 2 PO 4 ), 0.1-0.5wt %; sodium chloride (NaCl), 5.0-10.0 wt %; magnesium sulfate heptahydrate (MgSO 4 ⁇ 7H 2 O), 0.01-0.05 wt%; ammonium sulfate (NH 4 ) 2 SO 4 , 0.05 wt %-0.5wt % and peptone, 0.02-0.1 wt %.
  • the suitable medium for culturing is a synthetic medium with a pH value ranging from 6 to 8. In a more preferred embodiment, the suitable medium has a pH value of 7.
  • the culture step is conducted in fermenters operating at a temperature ranging from 25°C to 37°C. In a more preferred embodiment, the temperature is 32°C.
  • the present invention provides a fermentation process comprising a fermentation step allowing to grow a strain of the corresponding bacteria, preferably wherein said bacteria is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus, more preferably from genus Halomonas, still more preferably from Halomonas elongata sp., in a suitable medium in a fermenter under conditions of agitation sufficient to maintain a homogenous culture and limited aeration such that dissolved oxygen pressure (pO2) within the culture is around 20 to 40 % for most of the fermentation step.
  • pO2 within the culture is 30 % in the fermentation step.
  • the inventive process of preparing an exopolysaccharide-protein complex comprises the step of isolating the exopolysaccharide-protein complex from the secreted fraction of the culture.
  • Said isolation can be carried by removing other molecules present in the culture media by alcohol precipitation.
  • alcohols which can be used include ethanol, isopropanol, and methanol.
  • the isolation and purification of the EPCx after alcohol precipitation can be conducted by tangential flow filtration methods using ultrafiltration membranes.
  • said membranes have a MWCO of 30 kDa and the retentate recovered after the ultrafiltration comprises the exopolysaccharide and the EPS-associate proteins which can be recovered.
  • the tangential flow filtration can act to both diafilter and concentrate the EPCx.
  • the present invention relates to a composition (pharmaceutical, nutraceutical or cosmeceutical) comprising an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof.
  • said composition further comprises a biological response modifier selected from the group consisting of lymphokine, interleukin, growth factor and NFkB factor.
  • the present invention relates to a composition comprising an exopolysaccharide-protein complex according to the first object of the invention, including each of the embodiments comprised in said first aspect and combinations thereof, for use in stimulating an immune response in a subject for preventing or treating a disease selected from cancer or a disease associated to undesirable inflammatory activity.
  • said disease associated to undesirable inflammatory activity is selected from allergy, Alzheimer's disease, arthritis, autoimmune deficiency syndrome, celiac disease, diabetes mellitus, gastrointestinal disorder, inflammatory bowel disease, interstitial cystitis, skin disorders, acne, arteritis, arthritis, cancer, cellulitis, dermatitis and cardiovascular diseases.
  • said cancer is liver cancer or a hepatocellular carcinoma.
  • said exopolysaccharide-protein complex according to the first object of the invention is used as an anticancer agent or adjuvant agent for cancer therapies.
  • the present invention provides an in vitro toxicity test to evaluate the cytotoxic potential of EPCx on non-tumor cell lines.
  • the cytotoxicity evaluation of each concentration of EPCx was performed by using the MTT Cell Proliferation Assay in order to determine the concentrations which are not harmful to Human Epidermal Keratinocytes (HEK) and human monocyte-like cells (THP-1).
  • HEK Human Epidermal Keratinocytes
  • THP-1 human monocyte-like cells
  • the EPCx show in vitro anti-proliferative activity against a panel of one or more cancer cell lines, including human lung adenocarcinoma cell line (H1975), Human melanoma cell line (A375), and human hepatocellular carcinoma cell line (HepG2).
  • human lung adenocarcinoma cell line H1975)
  • Human melanoma cell line A375
  • human hepatocellular carcinoma cell line HepG2
  • the anticancer properties of EPCx was ascertained by MTT assay and showed that EPCx significantly inhibit the growth of human lung adenocarcinoma cell line (H1975), Human melanoma cell line (A375), and human hepatocellular carcinoma cell line (HepG2).
  • EPCx The immunomodulatory and anti-proliferative properties of EPCx were ascertained by the analysis of the induced activation of human primary keratinocytes (HEK) and human monocyte-like cells (THP-1) stressed with LPS (lipopolysaccharide), analyzing the expression of mRNA for the cytokines IL-6, IL-8, IL-10 and TNF- ⁇ by real-time PCR and the NF- ⁇ B activation using a luciferase reporter gene assay.
  • HEK human primary keratinocytes
  • TNF-1 human monocyte-like cells
  • LPS lipopolysaccharide
  • the immunomodulatory and anti-cancer properties of the EPCx are showed preferably at EPCx concentrations between 0.01 mg/ml to 1.0 mg/ml.
  • an EPCx obtained from a bacterium preferably wherein said bacterium is from a genus selected from Halomonas, Pseudoalteromonas, Vibrio, Salinivibrio, Marinomonas, Alteromonas, Pseudomonas, Halobacillus, and Bacillus, more preferably from genus Halomonas, still more preferably from Halomonas elongata sp., in accordance with the invention, can be used as anti-inflammatory and/or inmunomodulator agent, and is in particular able to inhibit a pro-inflammatory stimulation of normal cells in culture.
  • EPCx exhibits tumor anti-promoting properties in cancer cell lines culture.
  • Example 1 Isolation and Identification of the microorganism.
  • a microorganism included in the present invention is isolated from a rock salt from solar salternsin Cardona(Spain) by serial dilution and plating on MH agar medium(Ventosa, Garcia, Kamekura, Onishi, & Ruizberraquero, 1989). The plates were incubated at 32°C for 3 to 5 days, and bacterial colonies were isolated in pure form and maintained on slopes of the same medium.
  • the bacterium isolates was grown in MY medium (Moraine & Rogovin, 1966) supplemented with 5-10 % NaCl for 3-5 days at 32°C under continuous shaking (120 rpm).
  • the EPS from the growing culture was isolated using the method as described by Quesada et al. (Quesada, Bejar, & Calvo, 1993) and used for quantification and chemical analysis.
  • the selected microorganism was analyzed by physiological and biochemical methods following standard microbiological methods (Table 1). As a result, based on physiological and biochemical analysis thereof, the microorganism of the present invention was confirmed to have similarity to Halomonas genus. Table 1. Morphological and physiological characteristics of the isolated microorganism. Character Morphological characters Colony morphology Cream, circular Gram nature - Cell shape Rod Cell arrangement Single Motility + Pigmentation - Physiological characters - pH range for growth 5-11 pH optimum for growth 8 Temp. range for growth (°C) 22-40 Temp.
  • the present inventors analyzed base sequence of ribosome small subunit gene. Particularly, genomic DNA was separated by using MasterPure TM DNA purification from Epicentre ® Biotechnologies Germany according to the manufacturer's instruction. PCR mediated amplification of 16S rDNA and purification of the PCR product was carried out as described previously (Rainey, WardRainey, Kroppenstedt, & Stackebrandt, 1996). As a result of database analysis on the decided base sequence using NCBI (National Center for Biological Information) BLAST, the microorganism was confirmed to have similarity to Halomonas genera. However, it had a little difference from the typical Halomonas elongata, so that the microorganism of the present invention was finally named as Halomonas elongata.
  • NCBI National Center for Biological Information
  • the partial 16S rRNA gene sequence is shown in figure 5 .
  • Example 2 Preparation and Isolation of the Exopolysaccharide-Protein Complex (EPCx) excreted by Halomonas elongata according to example 1.
  • EPCx Exopolysaccharide-Protein Complex
  • the strain of the species Halomonas elongata was cultured in a fermenter, at 32°C and at a pH of 7.5, whose broth contained (g L -1 ): 100 NaCl; 50 Lactose; 7 K 2 HPO4; 2 KH 2 PO4; 0.1 MgSO4 ⁇ 7H 2 O; 1 (NH 4 ) 2 SO 4 and 0.5 Peptone.
  • An inoculum was prepared with 10% (v/v) of a pre-culture and the duration of the fermentation was extended to 72 hours.
  • the reactors are operated in batch mode, and dissolved oxygen was controlled by the agitation (300 to 900 rpm) at pO 2 ⁇ 30%.
  • the bacteria were separated from the broth by centrifugation at 12,000 g for 45 min.
  • the resulting clear solution was subjected to ultrafiltration and dialysis using an installation for ultrafiltration (Sartocon® Slice Cassette, Sartorius Stedim), membrane exclusion limit 30 KDa. If necessary, the final solution may be lyophilized and purified obtaining an exopolysaccharide-protein complex with a yield of 60-80%.
  • the content of neutral and acid monosaccharides of the exopolysaccharide obtained according to Example 2 was determined by a method described by Honda et al.(Honda et al., 1989) and Yang et al. (X. B. Yang, Zhao, Wang, Wang, & Mei, 2005; X. B. Yang, Zhao, Zhou, et al., 2005). Briefly, the purified polysaccharide sample (1 mg) was hydrolyzed with 1 ml of 2 M trifluoroacetic acid at 120°C for 2 h, derivatized with 1-phenyl-3-methyl-5-pyrazolone, and subsequently analyzed by high-performance liquid chromatography with detection by absorbance monitoring at 245 nm.
  • the percent relationship of sugars obtained was 30-60% of glucose, 30-50% galactose, 5-10% of glucuronic acid, 1-10% of rhamnose and 1-10% of glucosamine, being the amounts consistent with a total of 100%.
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970) was carried out with a 4% stacking and a 9% separating gel.
  • the EPCx sample was dissolved at 10 mg/mL in distilled water and added at 1:3 volume ratio into a buffer solution of 0.5% SDS with 1 %-mercaptoethanol, and then heated to boiling for 5 min.
  • the gels were stained with Coomassie Brilliant Blue R-250 to visualize proteins. 12 detectable bands, including three principal bands of 38 kDa, 46 kDa and 54 kDa were observed (see figure 1 ).
  • the cytotoxicity evaluation of exopolysaccharide-protein complex was performed by using the MTT assay in order to determine the concentrations which are not harmful to Human Epidermal Keratinocytes (HEK) and human monocyte-like cells (THP-1). Proliferation of cells lines was measured based on the mitochondria-dependent reduction of yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), to generate reducing equivalents such as NADH and NADPH. Briefly, 5 ⁇ 10 3 cells in 100 ⁇ L per well were plated in 96-well tissue culture plates for 24 h.
  • EPS EPS to modulate the inflammatory response was evaluated in vitro in human primary keratinocytes (HEK) and human monocyte-like cells (THP-1) stressed with LPS (lipopolysaccharide).
  • the cells were pretreated with different concentrations of exopolysaccharide-protein complex (1, 0.1 and 0.01 mg/ml) for 24 h and with 10 ⁇ g/ml of LPS for 24h.
  • Controls of HEK and THP-1 cells treated only with the different concentrations of exopolysaccharide-protein complex were also prepared.
  • mRNA for the cytokines IL-6, IL-8, IL-10 and TNF- ⁇ was analyzed by real-time PCR and the NF- ⁇ B activation was measured using a luciferase reporter gene assay.
  • CT threshold cycles
  • EPCx exopolysaccharide-protein complex
  • Gene expression (mRNA, real-time PCR) of pro-inflammatory cytokines (IL-6, IL-8, and TNF- ⁇ ) and anti-inflammatory cytokine (IL-10) in keratinocytes stresses with LPS and treated with different concentrations of EPCx (1.0, 0.1 and 0.01 mg/ml).
  • Gene expression (mRNA, real-time PCR) IL-6 IL-8 TNF- ⁇ IL-10 Control 3 2 4 1 LPS 62 59 67 7 EPCx 0.01 12 17 21 11 EPCx 0.1 17 14 27 18 EPCx 1.0 15 18 31 22 Table 4.
  • Gene expression of pro-inflammatory cytokines (IL-6, IL-8, and TNF- ⁇ ) and anti- inflammatory cytokine (IL-10) in human monocyte-like cells stresses with LPS and treated with different concentrations of EPCx (1, 0.1 and 0.01 mg/ml).
  • Gene expression IL-6 IL-8 TNF- ⁇ IL-10 Control 1.5 3 2 1 LPS 73 82 67 5 EPCx 0.01 21 27 32 12 EPCx 0.1 27 30 44 12 EPCx 1.0 15 18 31 15
  • NF- ⁇ B activation was measured using a luciferase reporter gene assay.
  • THP-1 macrophages (1 ⁇ 10 5 cells ⁇ well -1 ) were transfected with the pNF- ⁇ Bluciferase reporter gene construct (Stratagene) using Lipofectamine LTX plus (Invitrogen).
  • EPS 1,0.1 and 0.01 mg/ml
  • lysis buffer 200 ⁇ L ⁇ well -1
  • lysis buffer 25 mM glycylglycine, 15 mM MgSO 4 , 4 mM EGTA, 1 mM dithiothreitol and 1% Triton X-100.
  • Lysed cells were centrifuged (5 min, 9000 g) and stored at -80 °C until assay.
  • Luciferase activity was measured using luciferin (1 mM in glycylglycine buffer, 300 ⁇ L ⁇ sample -1 ) in a luminometer at 562 nm.
  • the MTT assay was used for measuring the proliferation of the tumor cells. Briefly, human hepatocellular carcinoma cell line (HepG2), human breast adenocarcinoma cell line (MCF7), human lung adenocarcinoma cell line (H1975) and Human melanoma cell line (A375) were seeded at a density of 4 ⁇ 10 4 cells/mL in a volume of 0.1 mL in 96-well plates, respectively. After 24 h, different concentrations of EPCx (1.0, 0.1 and 0.01 mg/ml) were dissolved in the medium was added to each well and incubated for 48 h at 37 °C in a CO 2 incubator. 5-Fu (5-fluorouracil) was used as the positive control.
  • EPCx 1.0, 0.1 and 0.01 mg/ml
  • EPCx markedly inhibited proliferation of human hepatocellular carcinoma cell line (HepG2) in a dose-dependent manner ( Figure 4 ), with little effect on growth of human breast adenocarcinoma cell line (MCF7), human lung adenocarcinoma cell line (H1975) and Human melanoma cell line (A375).
  • MCF7 human breast adenocarcinoma cell line
  • H1975 human lung adenocarcinoma cell line
  • A375 Human melanoma cell line
  • the potency of EPCx (at 1.0 mg/mL) to HepG2 cells was found to be similar to 5-fluorouracil (5-FU, IC50 was 10.0 ⁇ mol/L).

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